Characterization of the carboxyltransferase domain in acyl -CoA carboxylase (ACCase) from Streptomyces coelicolor and Mycobacterium tuberculosis

The building blocks for fatty acid and polyketide biosynthesis are provided by acyl-CoA carboxylase (ACCases), such as acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC) that carboxylate the α-carbon of acetyl- and propionyl-CoA to produce malonyl- and methylmalonyl-CoA, respectively. The development of ACCase-related therapeutics has been hampered by the lack of molecular information on how ACCases recognize their corresponding substrates or inhibitors. Presented herein are the crystallographic, mutagenesis and kinetic studies aimed at determining the molecular basis for substrate specificity of ACC and PCC from Streptomyces coelicolor. The crystal structures of seven PccB wild type and mutant crystal structures were compared systematically, enabling the establishment of a structure-function relationship of these PccB mutants with the observed substrate specificity towards acetyl-, propionyl- and butyryl-CoA. The above results allow us to dissect the molecular features responsible for substrate specificity of ACCases.;Mycolic acids and multimethyl-branched fatty acids are found uniquely in the cell envelop of pathogenic Mycobacteria, which are essential for the survival, virulence and antibiotic resistance of Mycobacterium tuberculosis. Acyl-CoA carboxylases (ACCase) commit acyl-CoAs to the biosynthesis of these unique fatty acids. Unlike other organisms, Mycobacterium tuberculosis contains six ACCase carboxyltransferase domains, AccD1-6, whose specific roles in the pathogen are not well defined. Previous studies indicate that AccD4, AccD5 and AccD6 are important for cell envelope lipid biosynthesis and that its disruption leads to pathogen death. We have determined the crystal structure of AccD5 and AccD6, whose sequences, structures, and active sites are highly conserved with respect to the carboxyltransferase domain of the Streptomyces coelicolor propionyl-CoA carboxylase (PccB). Both crystals structure and kinetic assays indicate that AccD5 prefers propionyl-CoA as its substrate and produces methylmalonyl-CoA, the extender unit for multimethyl-branched fatty acids. In contrast to AccD5, AccD6 prefers acetyl-CoA to generate malonyl-CoA, the extender unit for fatty acids and mycolic acids. Based on both structures, we conducted extensive in-silico screening based on compounds from the National Cancer Institute and the UC Irvine ChemDB database, and identified inhibitors NCI-65828 ( Ki=13.5 µM for AccD5) and NCI-172033 (K i=1.8 µM for AccD6). Moreover, NCI-172033 also inhibits the growth of several pathogenic species of Mycobacteria, including multidrug-resistant strains of M. tuberculosis, at low µM concentrations. The compound provides a strong inhibition of both fatty acid and mycolic acid biosynthesis at MICs concentrations. Our results pave the first step towards understanding the biological roles of key ACCases that commit acyl-CoAs to the biosynthesis of cell envelope fatty acids. In addition, the structures obtained provide a new target for the development of anti-tuberculosis therapeutics.